alpha-Crystallin and small heat-shock proteins (sHSP) show sequence similarity along a stretch of 100 amino acids referred to as the alpha-crystallin domain. The presence of this domain is associated with the ability of sHSP to assemble into oligomeric structures and with their function as molecular chaperones. The purpose of this work is to determine whether highly conserved sequences in the alpha-crystallin domain of human heat shock protein 27 (HSP 27) and alphaA-crystallin are involved in contacts between subunits in the oligomeric structure. Sequential single cysteine mutants were constructed along the sequence between residues 109 and 120 in alpha-crystallin and along the corresponding sequence in HSP 27. The mutants were purified, spin-labeled and analyzed by Electron Paramagnetic Resonance (EPR) spectroscopy. Analysis of the sequence-specific environment of the attached spin labels reveals a periodicity of two consistent with the presence of a beta-strand along the sequences. The EPR spectral line shapes indicate that in both proteins, spin labels on different monomers are brought into close proximity in the oligomeric structure suggesting that equivalent strands from different subunits are interacting along an interface. A similar pattern of interactions is obtained in a mixed oligomer of alphaA-crystallin and HSP 27. Our data indicate a similar quarternary structure for alphaA-crystalline and HSP 27. Because the studied sequence is highly conserved across the sHSP family, we propose that the oligomeric structure of these proteins involves a conserved two-fold isologous association.